In the field of medical diagnostics including oncology, the detection, identification, quantitation and characterization of cells of interest, such as cancer cells, through testing of biological specimens is an important aspect of diagnosis. Typically, a biological specimen such as bone marrow, lymph nodes, peripheral blood, cerebrospinal fluid, urine, effusions, fine needle aspirates, peripheral blood scrapings or other materials are prepared by staining the specimen to identify cells of interest. One method of cell specimen preparation is to react a specimen with a specific probe which can be a monoclonal antibody, a polyclonal antiserum, or a nucleic acid which is reactive with a component of the cells of interest, such as tumor cells. The reaction may be detected using an enzymatic reaction, such as alkaline phosphatase or glucose oxidase or peroxidase to convert a soluble colorless substrate to a colored insoluble precipitate, or by directly conjugating a dye to the probe.
For example, substances sometimes known as markers may exist in a person's blood as a result of some medically abnormal condition. These markers may exist for such conditions as preleukemic cancers, a trisomy 21 fetus, or other known conditions which cause markers to exist in one's blood. These markers are normally not visually detectable. However, a blood cell sample may be fixed and bound with a substrate of an enzyme to produce a colored insoluble precipitate to identify the marker. The slides containing the prepared cellular specimens are then examined to evaluate the amount of the precipitate contained in the cellular specimen to determine whether the cellular specimen indicates that the condition exists in the person from which the sample was obtained.
For example, blood cells are classified into two types--red and white cells. The red cells carry oxygen in the form of hemoglobin to tissue in a person. The white cells are generally related to a person's immunity system. White blood cells are comprised of five types of which one, neutrophils, has a lobed nucleus which is typically used to identify this type of white blood cells. In response to the presence of a fetus, the neutrophil cells in the blood of a pregnant woman have an elevated level of alkaline phosphatase. If the fetus is a trisomy 21 fetus, the level of alkaline phosphatase in the neutrophils is even higher. Thus, the amount of alkaline phosphatase in the neutrophils of a pregnant woman's blood may be used as a marker for a trisomy 21 fetus. By preparing a blood specimen from a pregnant woman with a stain for identifying the alkaline phosphatase in neutrophils and a counterstain to facilitate detection of the lobed shaped nuclei of neutrophils, a pathologist may visually determine the likelihood that the fetus is a trisomy 21 fetus.
Examination of biological specimens in the past has been performed manually by either a lab technologist or a pathologist. In the manual method, a slide prepared with a biological specimen is viewed at a low magnification under a microscope to visually locate candidate cells of interest. Those areas of the slide where cells of interest are located are then viewed at a higher magnification to confirm those objects as cells of interest. The manual method is time consuming and may be susceptible to error including missing areas of the slide. In the example given above, the low magnification scan is performed to identify the neutrophils by the lobed shaped nuclei having the counterstain color.
Automated cell analysis systems have been developed to improve the speed and accuracy of the slide evaluation process. One known interactive system includes a single high power microscope objective for scanning a rack of slides, portions of which have been previously identified for assay by an operator. In that system, the operator first scans each slide at a low magnification similar to the manual method and notes the points of interest on the slide for later analysis. The operator then stores the address of the noted location and the associated function in a data file. Once the points of interest have been located and stored by the operator, the slide is then positioned in an automated analysis apparatus which acquires images of the slide at the marked points and performs an image analysis.
There are also known automated specimen analysis systems which automatically view slides located in carriers which are loaded in a hopper. The carriers are moved, one at a time, from the hopper to a motorized XY stage of the microscope. The motorized stage is operated to place one slide in the carrier under the objective turret of the microscope and the slide is scanned at a low magnification power. The view of the slide through the oculars of the microscope is captured by a camera which may either be a digital camera or an analog camera with an analog/digital (A/D) converter. The digitized image is provided to a computer subsystem coupled to the automated microscope to detect candidate objects of interest on the slide. The information regarding the candidate objects of interest is then stored and the viewing power for the objective turret is increased to a high magnification level. The slide is scanned at high magnification and an image of the slide at the high magnification level is captured by the camera, digitized, and further processed by the computer subsystem to eliminate debris and other objects which may be part of the cellular specimen which do not require analysis. The portions of the high magnification image which correspond to candidate objects of interest not eliminated by the processing at the high magnification level are then stored in a montage for viewing by a pathologist. Information identifying the slide from which the image was obtained and the location of the candidate object of interest on the slide is also recorded with the montage. If the pathologist wants to view the candidate object of interest on the slide, the pathologist may place the slide in a carrier and load it in the automated analysis system. The system then moves the slide to a position underneath the objective turret where, under high magnification, the pathologist may view the candidate object of interest to confirm the selection of the candidate object of interest for the montage.
One problem with previously known automated systems is the difficulty in evaluating the amount of a marker present in a cellular specimen. For example, neutrophil alkaline phosphatase (NAP) is typically stained to cause the insoluble product which identifies the marker to become red in color. The cellular specimen is usually counterstained to make the nuclei of the cells become blue in color. A pathologist viewing such a slide detects neutrophils by locating those cells which have a nucleus of the color, shape, and size expected for a neutrophil. The pathologist then subjectively evaluates the intensity of the red color for each located neutrophil and assigns a score to each one. The pathologist then subjectively determines whether the number of intensely red neutrophils and moderately red neutrophils are sufficient to conclude that the cellular specimen is indicative of a particular condition. For NAP, the pathologist usually grades the neutrophils with a rating of 0, 1, 2, 3, or 4 in accordance with a grading scale such as the one provided with the procedure for using the reagent kit sold by Sigma Diagnostics for demonstrating alkaline phosphatase activity in leukocytes. According to that procedure, a pathologist sums the subjectively assigned ratings for marker identifying precipitate in the first 100 neutrophils to arrive at a score which may be used to determine the relative red intensity of the neutrophils in the cellular sample. This score may then be used to determine whether the condition associated with the presence of the marker is indicated.
Previously known automated specimen analysis systems have not been able to provide grading as reliable as that possible with a trained pathologist. For example, U.S. Pat. No. 5,352,613 to Tafas et al. discloses a cytological screening method which purports to evaluate the presence and amount of such markers as NAP. However, the system of this patent has a number of limitations. For one, the perimeter of neutrophils or other candidate objects of interest must be precisely located in the images as the method of this patent computes an average optical density for each pixel within a candidate object of interest. However, where a candidate object of interest, such as a neutrophil, is overlapped by a red blood cell, the method of this patent may inaccurately define the perimeter of the candidate object of interest if it is using the red color component to define the perimeter for the candidate object of interest. When the perimeter of the candidate object of interest is not accurately defined, pixels actually in a candidate object of interest may be missed and those not actually in a candidate object of interest may be included in the computation of the density value. In fact, this patent does not indicate that the pixels for the nuclei of the cells being evaluated are excluded from the optical density measurements. In some cases, the inclusion of the nuclei pixels or exclusion of pixels actually in a cell may skew the measurements. Additionally, the method of this patent operates on a single color component of the image of the cellular specimen and, as a result, where cells may overlap, the pixel value of the single color component being analyzed may be attenuated in the light passed by the overlapped cells.
What is needed is a system which grades neutrophils containing NAP as reliably as a trained pathologist. What is needed is an automated specimen analysis system which does not rely on precise perimeter definition of candidate objects of interest in order to measure the amount of a marker identifying precipitate within a candidate object of interest. What is needed is a system which can accurately evaluate the amount of a marker identifying precipitate within a cellular specimen even though overlapping cells are present in the defined object of interest.